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Kentaro Kitahara and Shogo Matsumoto

An S-allele cDNA was cloned from pistils of 'McIntosh' apple (Malus ×domestica Borkh.). The allele, designated Si in Japan and S10 in Europe, is an S-RNase that is very similar (94%) to the S3-RNase at the deduced amino acid sequence level. This allele can be detected by amplification using the polymerase chain reaction (PCR) and specific primers, followed by digestion with restriction enzyme EheI. The S10 allele was discovered in 'Empire', 'Maypole', 'Shinano Red', 'Spencer', and 'Vista Bella'. The S-allele cDNAs sequenced to date are listed with their Japanese and European designations.

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Shogo Matsumoto and Kentaro Kitahara

A polymerase chain reaction (PCR)-based method for identifying the S-alleles in the Asian pear [Pyrus pyrifolia (Burm) Nak.] was applied to apple (Malus ×domestica Borkh.) cultivars. With minor modifications in one of the primers, the fragments from S-genes (S-RNases) with introns were amplified from total DNA of apple cultivars possessing S2-, S3-, S5-, S7-(=Sd-), S9-(=Sc-), Sf- and Sg-allele genotypes. S-genes within S24-(=Sh-) and S26-alleles were also amplified. The PCR amplification step of this method appears to be useful for preliminary investigation of apple S-genotypes, especially for species or cultivars of unknown origin or history. Using the primers, which are a part of a new S-allele, the Se-allele encoding Se-RNase with an intron in the Se-allele was amplified. We cloned the cDNA of Se-RNase, and developed a PCR-restriction fragment length polymorphism (RFLP) analysis method for Se-allele identification. S-allele genotypes of seven apple cultivars were investigated.

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Ryoichi Nakajima, Shungo Otagaki, Katsuhiro Shiratake and Shogo Matsumoto

Previously, we showed that a reduction of FaTFL2 (homolog of Arabidopsis thaliana TFL1) expression is a key signal for flowering in domesticated octaploid strawberries (Fragaria ×ananassa). Since FaTFL2 expression seemed to be regulated by temperature rather than by photoperiod, we investigated whether down regulation of FaTFL2 and floral meristem generation occurred at different temperature conditions. In addition to the conditions for a normal super-forcing cultivation system of an 8-hour photoperiod and day/night temperatures of 31.2 or 30/15 °C, flowering also was generated under the same photoperiod and day/night temperatures of day/night-half/night-half temperatures of 30/15/25 °C conditions. We demonstrate that the new super-forcing cultivation system is energy saving based on the reduction of FaTFL2 expression.

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Hitomi Umemura, Katsuhiro Shiratake, Shogo Matsumoto, Tsutomu Maejima and Hiromitsu Komatsu

We re-investigated the flesh color and S-genotypes of progenies of red-fleshed apple cultivar JPP35, which was produced by ‘Jonathan’ × ‘Pink Pearl’, and clarified that 100% and 96% of progenies from ‘Shinano Sweet’ (S1S7) × ‘JPP35’ (S3S7) and ‘Orin’ (S2S7) × ‘JPP35’ (S3S7) containing S3-RNase allele, respectively, showed the red flesh trait. Using this tight linkage between red flesh trait and self- and cross-compatibility relating allele such as S3-RNase allele, we showed suitable cultivar combinations for efficient production of various red-fleshed apples. We also identified an unknown S-RNase allele in ‘Pink Pearl’ as S11 and determined its partial genomic sequence, including a complete intron with its known S3-RNase allele.

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Kentaro Kitahara, Junichi Soejima, Hiromitsu Komatsu, Hirokazu Fukui and Shogo Matsumoto

The S-locus genes in the pistil (S-RNases) were cloned from the apple (Malus ×domestica Borkh.) cultivar Akane (S-genotype SdSh from pollination analysis). The Sd- and Sh-RNase corresponded to S7- and S24-RNase, which have been cloned from `Idared' and `Braeburn', respectively. Sh-RNase was very similar to Sf- and Sg-RNases at the deduced amino acid-sequence levels (93%). We developed an S-allele specific polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis method for distinguishing the Sh from Sf and Sg, and the Sh-alleles of `Akane', `Touhoku 2', `Vista Bella', and `Worcester Pearmain' were identified. We also identified the S-allele genotypes of 16 apple cultivars.

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Shogo Matsumoto, Kentaro Kitahara, Sadao Komori and Junichi Soejima

S-allele genotypes of nine apple (Malus ×domestica Borkh.) cultivars were identified using S-allele–specific polymerase chain reaction (PCR)–restriction fragmentlength polymorphism (RFLP) analysis. A new S-allele, Sg, was proposed to be present in `American Summer Pearmain', `Indo', `Kitanosachi', and `Meku 10'. This allele is very similar to Sf at the nucleotide sequence (92%) and deduced amino acid sequence (94%) levels.

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Kentaro Kitahara, Shogo Matsumoto, Toshiya Yamamoto, Junichi Soejima, Tetsuya Kimura, Hiromitsu Komatsu and Kazuyuki Abe

As the parents of the some of the apple cultivars were unknown and others were uncertain, we investigated the parent-offspring relationships of eight apple cultivars by S-RNase analysis and SSR markers. The paternal parent of `Hida' was identified as `Golden Delicious', not the previously mentioned `Orin'. It was indicated that `Ryoka No Kisetsu' and `Korin' showing identical SSR genotype are likely sports of `Fuji'. `Fuji', rather than `Toko', seemed to be a maternal parent of `Kotoku', but was not a paternal parent of `Orei', `Starking Delicious', `Nero 26', `Empire', or `Aori 3'. Previously mentioned `Mutsu', `Indo', and `Shin Indo' were excluded as paternal parents of `Hokuto'. `Tsugaru' and `Jonathan' and were identified as the respective paternal parents of three cultivars described as having unknown paternal parents, i.e., `Aika No Kaori', `Yoko', and `Tsugaru'.

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Keiko Sekido, Yusaku Hayashi, Kunio Yamada, Katsuhiro Shiratake, Shogo Matsumoto, Tsutomu Maejima and Hiromitsu Komatsu

We have used a red-fleshed apple cultivar, Malus ×domestica Pink Pearl, and its progeny, ‘JPP 35’, as paternal parents for producing new red-fleshed cultivars suitable for fresh use or processing such as pie fillings, dried apple, apple juice, or cider. In this process, we found that the S3-RNase allele of ‘Pink Pearl’ was linked to its red flesh trait. It was suggested that this trait might be controlled by a new gene apart from the MYB10 (MdMYB10) gene. Using ‘JPP 35’ (S-RNase allele genotype; S3S7) produced by ‘Jonathan’ (S7S9) × ‘Pink Pearl’ (S3Sx) as a paternal parent, we developed a system for producing red-fleshed progenies suitable for fresh use. That is, 96% and 86% of progenies from ‘Shinano Sweet’ (S1S7) × ‘JPP35’ (S3S7) and ‘Orin’ (S2S7) × ‘JPP35’ (S3S7) containing the S3-RNase allele, respectively, showed the red flesh trait. Similarly, red-fleshed progenies suitable for apple pie or natural red juice could be produced by ‘Jonathan’ (S7S9) × ‘JPP35’ (S3S7).

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Hiroaki Ito, Masaki Ochiai, Hiroaki Kato, Katsuhiro Shiratake, Daigo Takemoto, Shungo Otagaki and Shogo Matsumoto

We have succeeded in establishing a virus-induced gene silencing (VIGS) of rose using Apple latent spherical virus (ALSV) vectors. An ALSV infection on rose did not cause any symptoms like those observed on other plant species and grew healthy. We have cloned and sequenced the phytoene desaturase (PDS) gene in wild rose, then used its fragment for silencing the rose internal PDS gene. The silencing phenotypes such as the highly uniform photo-bleached phenotype with PDS inhibitions were observed on the upper leaves of primary shoots and on a secondary shoot of R. rugosa for more than 5 months. ALSV vectors seemed useful for analyzing gene function and for the molecular breeding of rose.

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Kentaro Kitahara, Shogo Matsumoto, Toshiya Yamamoto, Junichi Soejima, Tetsuya Kimura, Hiromitsu Komatsu and Kazuyuki Abe

We examined the genetic diversity and relatedness among apple (Malus ×domestica Borkh.) cultivars in Japan. The 42 apple cultivars, including major cultivars in Japan, were divided into five groups based on SSR genotypes. Most economically important cultivars belong in three groups: Fuji-Delicious, Golden Delicious, and Jonathan groups, and their genetic backgrounds seemed to be narrow. We also investigated the parent-offspring relationships of nine apple cultivars. `Jonathan', `Fuji', and `Rero 11' were identified as the respective paternal parents of three cultivars described as having unknown paternal parents (i.e., `Akagi', `Ambitious', and `Hokuto'). `Starking Delicious', `Senshu', and `Golden Delicious', rather than `Ralls Janet', `Hatsuaki', and `Indo', seemed to be the paternal parents of `Kinsei', `Kiou', and `Mellow', respectively. `Carolina Red June' was excluded as a paternal parent of `Ranzan'. Both attributed parents of `Scarlet' (`Akane' and `Starking Delicious') were excluded, and it was suggested that `Fuji' was used as either a maternal or a paternal parent of `Scarlet'. `Jonathan' rather than `McIntosh' seems to be a maternal parent of `Yukari'.